The striking concentration of Na+ channels at the node results from complex interactions between axons and myelinating glial cells, i.e. Schwann cells in the PNS and oligodendrocytes in the CNS. The nature of these interactions are poorly understood as are the mechanisms by which Na+ channels are targeted to and assemble into a larger complex with other proteins at the node. Associated proteins include the 186 kD isoform of neurofascin, which may bind to receptors on overlying glial processes, and ankyrin G which links cell adhesion molecules to Na+ channels. The node is flanked by paranodal junctions, which are dispensible for initial node formation, but may regulate the maturation and channel density at the node. We propose to address important features of this model by characterizing the targeting of proteins to the node, the role of glial processes that overlie and flank the node and the potential role of neurofascin in directing assembly of the node. Specifically, we will i) determine whether proteins targeted to the node redistribute from existing pools and/or are newly synthesized and transported to this site by labeling live cocultures of neurons with anti-NrCAM Fab fragments as they undergo myelination, analyzing whether nodes form after transecting axons of the Wlds/Ola mouse, and characterizing axonal transport of Na+ channels and other proteins to the node; ii) investigate the role of the ERM+ Schwann cell processes in PNS node formation by blocking their formation by dominant negative strategies or the use of Rho kinase inhibitors; iii) characterize the role of the flanking paranodal processes and junctions in the development and maturation of the node in Caspr deficient mice which lack paranodal junctions focusing on node width, channel density and Na+ channel subtypes and iv) determine the role of the L1 family of cell adhesion molecules, in particular neurofascin, in node formation by generating mice with a conditional, neuron-specific knockout of neurofascin and determine whether they exhibit aberrant initial segment, node or paranode organization and, if minimally affected, by crossing these mice to an existing line of NrCAM knockout mice for analysis of the double knockouts. These studies should provide important new insights into the mechanisms responsible for the assembly of the node, the nature of the glial signals that direct its assembly and may clarify the functional deficits that accompany demyelinating disorders and interrupt normal saltatory conduction.